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Maintenance and modulation of T cell polarity - Departments of ...

REVIEW

© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

Maintenance and modulation of

T cell polarity

Matthew F Krummel 1 & Ian Macara 2

As T cells move through the lymphatics and tissues, chemokine receptors, adhesion molecules, costimulatory molecules and

antigen receptors engage their ligands in the microenvironment and contribute to establishing and maintaining cell polarity.

Cytoskeletal assemblies, surface proteins and vesicle traffic are essential components of polarity and probably stabilize the

activity of lymphocytes that must negotiate their ‘noisy’ environment. An additional component of polarity is a family of polarity

proteins in T cells that includes Dlg, Scrib and Lgl, as well as a complex of partitioning-defective proteins. Ultimately, the

strength of a T cell response may rely on correct T cell polarization. Therefore, loss of polarity regulators or guidance cues may

interfere with T cell activation.

T cells traffic through a signal-rich environment. In the blood, they receive

cues from chemokines, which permit attachment and extravasation into

tissues. In secondary lymphoid environments, receptors on the T cell surface

integrate signals from ligands bound to other cells or to the extracellular

matrix and from gradients of soluble mediators. At the sites of effector

function, T cells encounter secreted products of activated macrophages,

neutrophils and natural killer cells, which may result in polarized or nonpolarized

stimulation of cytokine receptors on T cells. Underlying a successful

T cell response, then, is the ability to ‘prioritize’ these cues and subsequently

orient effector mechanisms toward the optimal targets. This review discusses

polarity ‘themes’ in T cells and their potential inter-relationships

and will postulate how polarity is modulated when T cell receptor (TCR)

stimuli are encountered.

Polarity ‘themes’ in motile lymphocytes

Like a musical partita, the five systems of T cell morphology are variations

on a shared polarity ‘theme’. The actin cytoskeleton, tubulin cytoskeleton,

surface proteins, vesicle traffic and conserved polarity proteins are all polarized

relative to the motility axis. Among the best studied of those systems are

the actomyosin cytoskeleton and the GTPases associated with promoting

actin nucleation (Fig. 1a). Actin fibers are inherently polar fibers with their

barbed (‘plus’) end at the site of monomer addition and in the direction of

actin-based protrusion. That constitutes the leading edge in lymphocytes

and is probably very similar to the lammellipodial regions generated in

other motile cell types, in that free actin filaments drive protrusion independently

of myosin II crosslinking activities 1 . However, behind the leading

edge and extending through the midbody is a zone of high myosin II

1 Department of Pathology, University of California at San Francisco, San

Francisco, California 94143-0511, USA. 2 Center for Cell Signaling, Department

of Microbiology, University of Virginia School of Medicine, Charlottesville,

Virginia 22908-0577, USA. Correspondence should be addressed to M.F.K.

(matthew.krummel@ucsf.edu) or I.M. (igm9c@virginia.edu).

Received 7 August; accepted 14 September; published online 19 October

2006; doi:10.1038/ni1404

accumulation 2 in which it is likely that actin is organized by myosin in

rods that are mostly parallel. In epithelial cells, this region corresponds to

the lamella, and the myosin II in this zone causes retrograde flow of actin

filaments and attached membranes toward the rear of the cell 1 . Rearward

movement of surface-bound beads toward the uropod has demonstrated

that retrograde cortical flow also occurs in T cells 3 . Myosin II ‘motors’ are

probably responsible for retrograde flow, as shown by the accumulation of

myosin II at sites of leading edge retraction and the subsequent retrograde

flow of those motors into the uropod 2 .

The microtubule cytoskeleton and related kinesin and dynein motors

comprise a second polarized cytoskeleton (Fig. 1b). Like actin filaments,

microtubules are inherently polarized. Microtubules are organized around

a microtubule-organizing center (MTOC). That arrangement creates

another level of polarity, as the MTOC is invariably in the uropod and

behind the nucleus.

Surface receptors and perhaps membrane lipid rafts are also polarized

such that some are localized toward the uropod, whereas others, activated

integrins in particular, are likely to cycle toward and accumulate at the leading

edge 4 (Fig. 1c). Actomyosin and membrane movement from the front

of the cell to the rear of the cell probably accounts for the general accumulation

of many surface receptors at the uropod: TCRs, CD2, CD43 and CD44

accumulate at the uropod before activation 5–8 ; ICAM, CD43 and CD44

in particular are probably localized as a result of their association with

the C terminus–phosphorylated form of ezrin-radixin-moesin (cpERM),

which associates with actin 5,6 . In comparison, adhesive receptors such as

LFA-1 are selectively retained along the ‘midbody’ of the cell by adhesion 4 .

Cholesterol-rich rafts may also be polarized along with those receptors 9 .

Integrins may also be specifically recycled to the leading edge by as-yetunidentified

selective vesicular traffic from the uropod to the leading edge

(Fig. 1d). Cycling is important, as forward motility requires the internalization

of highly adhesive integrins at the front and midbody at the uropod

and that they be recycled ‘forward’ to contribute to new projections. It is

highly probable that endocytosis and exocytosis are generally polarized

along the axis of movement and also reinforce other systems of polarity

by asymmetrically depositing material at one end of the cell or the other.

NATURE IMMUNOLOGY VOLUME 7 NUMBER 11 NOVEMBER 2006 1143


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a b c d e

Actomyosin cytoskeleton

Microtubule cytoskeleton

Surface receptors

Vesicle traffic

Polarity proteins

© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

Uropodal: Lamella:

contraction actomyosin

and dissembly retrograde

flow

+

+

Rac

cdc42

+

+

+

Lamellapodia:

assembly

and branching

cpERM

ERM-bound

Unengaged receptors

Adhesive integrin

Much vesicle movement probably involves the Rab family of proteins that

direct the fusion of vesicles with the plasma membrane and also associate

with specific myosins or kinesins for directional transport. The targeting

and docking of vesicles with membranes involves the recognition of vesicle

soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-

SNAREs) by target SNAREs (t-SNAREs) that are enriched at specific sites

along the membrane. SNAREs typically consist of syntaxin and SNAP proteins,

some of which have been identified in T lymphocytes 10,11 . Polarized

distribution of t-SNAREs at the leading edge of motile T cells remains to

be assessed, but would be predicted to ‘encourage’ delivery of vesicles in

the direction of motility.

The proteins scribbled (Scrib), lethal giant larvae (Lgl) and discs

large (Dlg), together with partitioning-defective (PAR) polarity proteins

(Fig. 1e), have emerged as a fifth set of participants regulating many aspects

of lymphocyte biology. Scrib, Lgl and Dlg proteins were first identified by

genetic screens in flies or worms and are key conserved elements required

for polarization of cells in many contexts. For example, Scrib was identified

in a screen for genes involved in apical versus basal polarity in drosophila

embryonic epithelium, but it also functions in synaptic vesicle dynamics 12

and acts as a tumor suppressor 13 . Scrib interacts genetically with the two

other tumor suppressors Lgl and Dlg, which are also essential for maintaining

apical versus basal polarity. Somewhat unexpectedly, no existing

evidence indicates the involvement of mammalian Scrib protein in the

maintenance of apical versus basal polarity, but Scrib has been linked to

the regulation of planar polarity of the inner ear epithelium 14 , in epithelial

cell-cell adhesion 15 , in endocytosis and recycling of G protein–coupled

receptors (GPCRs) 16 and, most unexpectedly, in T cell polarization 17 .

PAR proteins were first identified in a screen for genes affecting the first

asymmetric division of the Caenorhabditis elegans zygote. Of the six PAR

genes that have been cloned, those encoding PAR-3, PAR-6 and atypical

protein kinase C (aPKC) interact both genetically and physically. PAR-6

seems to function at least in part as a targeting subunit for aPKC, to which it

binds constitutively. The PAR proteins also interact with Scrib, Lgl and Dlg

proteins. For example, PAR-6 can recruit Lgl, which is then phosphorylated

by aPKC 18–20 .

Polarized assemblies in motile lymphocytes

T cells have a directional axis of motility, and a fundamental driving force

for the polarization and migration of T cells is the dynamic remodeling

of actin cytoskeletal elements. In most cell types that have been studied,

remodeling is controlled by the Rho family of GTPases through a plethora

of effector proteins. Rac and Cdc42 GTPases, in particular, have been associated

with the protrusion of the leading edge and in the orientation of

Endocytosis

Figure 1 Five polarity systems in motile T lymphocytes. (a,b) Polarity can be described from the perspective of the actomyosin cytoskeleton (a) or tubulin

cytoskeleton (b), which are polarized relative to the direction of migration (green arrow). (c) A front-to-back ‘footprint’ of integrins on substrates (red shading)

and uropodal accumulation of receptors further delineates the polarity. (d) Both directional migration and the associated cycling of integrins require the

movement of material through the cytoplasm by means of an endocytic-exocytic loop. (e) Evidence places evolutionarily conserved polarity proteins in distinct

locales during migration.

Exocytosis

Scrib

Dlg

Crumbs

Lgl

PAR-3

migration (Fig. 2a). Overexpression of mutant forms of either GTPase

results in loss of polarity and defective chemotaxis toward the chemokine

SDF-1 (refs. 21,22). ‘Downstream’ effectors of Rac and/or Cdc42 include

the Wiskott-Aldrich syndrome protein (WASP), neural WASP and the

related WAVE (also called Scar) family of proteins, which induce actin

nucleation through the complex of Arp2 and Arp3. WASP is required for

optimal chemotaxis 23,24 , but evidence has indicated WAVE2 is a predominant

participant in hematopoietic cell migration 25–27 . WAVE2 is required

for actin cap formation at the immunological synapse (IS) and forms a

complex with the Hem-1 linker protein 26 . Hem-1 is required for actin

protrusions in neutrophils and augments leading-edge identity by inhibiting

activation of the myosin light chain at that site 25 .

In contrast to Rac and Cdc42, which promote actin nucleation, the

GTPase RhoA activates a protein kinase, ROCK, that phosphorylates

myosin light chains and thus increases the contraction of actin filaments

relative to each other (Fig. 2b). Rho is ‘downstream’ of chemokine GPCRs

and is required for increases in integrin affinity 28,29 . Thus, it is likely that

in contrast to Rac and Cdc42, Rho proteins located at the site of GPCR

signaling activate myosin, which triggers sliding of this membrane patch

toward the cell uropod, thereby generating forward thrust in the rest of the

cell. Although that model is likely, the system is more complex locally, as

a Rac complex with its effector PAK have also been linked to the chemotaxis

of both macrophages and T cells 30,31 . As Rac often antagonizes Rho

function 32 , the details of how these GTPases function in concert remains

a mystery.

In T cells, Rho is also required for uropod formation, possibly because

of its involvement in augmenting phosphorylation of ERM 33 . That function

is important, because cpERM in the uropod may tether a complex

that aids in recycling components back to the leading edge through vesicle

trafficking mediated by ADP-ribosylation factor (Arf; Fig. 2c). Arf family

GTPases control vesicle movement during motility in autologous systems 34 ,

although the function of these proteins in T cells has not been established.

The PDZ domains of mammalian Scrib associate with the C terminus of

the Rac guanine-exchange factor βPIX 35 , which in turn binds to GIT1,

a scaffold protein with an Arf GTPase-activating protein domain. This

Scrib-βPIX-GIT1 complex has been linked to the recycling of GPCRs 16 in

autologous systems, suggesting a similar function in T cells. Scrib is localized

in the uropod 17 , but how does that localization occur? In drosophila

neurons, Dlg determines the localization of Scrib, and these two proteins

form a complex that is mediated via the scaffold protein ‘GUK-holder’ 36 .

A mammalian homolog of GUK-holder has been identified, but it has

not yet been shown to be functionally equivalent to the fly protein, and

its expression in T cells is untested. However, human Dlg is targeted to

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© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

membranes by its association with ERM 37 , and Dlg proteins are found in

the T cell uropod alongside cpERM proteins 17 . Notably, Scrib-deficient

T cells fail to maintain polarity and cease movement, consistent with requisite

involvement of these proteins in a motility cycle 17 .

A final functional cassette is probably in the motile cell and consists of

PAR-3, Lgl, PAR-6 and aPKC-ζ which are all localized in the midbody 17 .

In epithelial cells, Lgl can associate not only with myosin II and PAR-6

but also with syntaxins, which control vesicle delivery to specific target

membranes. As mentioned above, PAR-6 seems to function at least in part

as a targeting subunit for aPKC, to which it binds constitutively. Thus, in

drosophila embryonic epithelial cells, PAR-6 can recruit Lgl, which is then

phosphorylated by aPKC 18–20 , which in turn triggers disassociation of the

complex from the cell cortex. The PAR-6–aPKC complex can also bind to

and phosphorylate the ubiquitin E3 ligase Smurf1, one target of which

is RhoA 38 . A key function of the PAR-6 polarity cassette might therefore

be to trigger local degradation of RhoA, which would reduce actin contractility

and block Rho-induced phosphorylation of cpERM proteins. In

addition, PAR-6–aPKC binds to and phosphorylates PAR-3 (refs. 39–41).

As PAR-3 is capable of binding to and spatially restricting the activity of

the Rac guanine-exchange factor Tiam1, the PAR-6–aPKC–PAR-3 interaction

probably also regulates actin dynamics 42,43 . Tiam1 was first identified

as a T cell lymphoma invasion and metastasis gene 44 . The association of

PAR-3 with LIM kinase (LIMK) may further regulate actin in the area

through LIMK regulation of cofilin 43 . Variations in the composition of

PAR-6–aPKC complexes may create unique actin dynamics in the midbody

that facilitate both forward motility through actomyosin regulation and

vesicle trafficking through syntaxin regulation (Fig. 2d). The nature of

those complexes remain to be elucidated.

Cytoskeletal polarity in the IS

In a signal-rich environment, interrelated feedback systems like those

described above are important for stabilizing cellular activity. In contrast,

TCR signaling initiates a transformation from high motility to the stable

IS 45,46 . How might that signal create the conditions in which proteins are

repolarized, and how is a stable IS maintained?

The reorientation of the T cell during engagement with an antigen-presenting

cell (APC) and formation of the IS are reminiscent of the switch

in polarization that occurs during the epithelialization of mesenchymal

cells. In both cases, cells undergo substantial morphological changes, which

are driven by remodeling of the actin and microtubule cytoskeletons and

by relocalization of polarity proteins. During the mesenchymal-epithelial

transition, anterior-posterior polarity is converted into apical-basal

polarity. Microtubules and actin reorganize to form bands around the cell

periphery, the Rac GTPase redistributes from the leading edge to the lateral

membrane, and polarity protein such as PAR-3, Scrib, and Dlg translocate

to the cell-cell junctions. A functionally similar switch occurs in T cells:

the uropod at the posterior of the T cell disappears 46 and polarity proteins

such as Scrib and Dlg transiently redistribute from the uropod to the front

of the cell where the IS forms 17,47,48 .

A first step in this transformation may be the disassembly of existing

cytoskeletal structures (Fig. 3a). TCR signaling transiently leads to the

dephosphorylation of cpERM, and that modification results in cytoskeletal

relaxation, probably because of release of the membrane-actin linkage

anchored by ERM 49 . That occurs through Rac activation 49 , which may

antagonize Rho-induced ERM phosphorylation 33 . Such relaxation may

release cell surface receptors and proteins such as Dlg and Scrib, which can

then traffic to the leading edge. TCR signaling also induces the calciumdependent

phosphorylation of myosin II (ref. 2). Myosin heavy-chain

phosphorylation results in loss of myosin filamentation 50 , probably releasing

the aligned actin filaments in the midbody and uropod and permitting

reorganization of the components formerly located in those regions.

At the leading edge, TCR signaling induces actin complexes and the early

assembly of microclusters of TCRs 7,51 . Microclusters probably arise as a

result of TCR-induced activation of the Lat and SLP-76 adaptor proteins,

which trigger WASP and PAK activation, which promotes actin assembly

52–54 (Fig. 3b). However, Dlg, which is found at the center of the contact,

c

d

b

a

a b c d

Leading-edge protrusion

Adherent contractile zone Uropodal recycling

Regulated midbody cytoskeleton

Hem-1

Myosin II

WASP

WAVE2

N-WASP

Actin

Rac

ROCK

Rho

MLC P

Rac

Myosin II

GPCR

LFA-1

Chemokine

ICAM

Dlg

cpERM

GUK-holder

Scrib βPIX

Arf GIT

GPCR

Syntaxin Myosin II

Lgl

PAR-6/aPKC or

PAR-3

LIMK Tiam1

Figure 2 Distinct zones in motile lymphocytes. Various functional compartments serve to stabilize crawling activity. All these molecules have been found

in the locations here except those in gray italics, which have not yet been assessed in T cells. (a) A protrusive complex that uses a WASP family member to

activate actin elongation is probably associated with the Hem-1 molecule in a cassette, which inhibits myosin light-chain activation. N-WASP, neural WASP.

(b) A contractile cassette ‘downstream’ of chemokine receptors and integrins may use Rho to activate ROCK and thereby induce myosin II contractility.

Additional signaling (such as via phosphatidylinositol-3-OH kinase) is not presented here. MLC, myosin light chain. (c) The uropod is proposed to be the site

of a cpERM-based internalization ‘engine’. The cpERM-mediated recruitment of Dlg may also recruit Scrib via a GUK-holder (not yet identified in T cells).

Scrib localization in the uropod in turn may recruit and activate Arf proteins via the assembly of βPIX-GIT complexes. (d) PAR-6–aPKC–based cassettes

throughout the cytoplasm may prevent actin elongation through PAR-3-mediated binding of LIMK or Tiam1. Alternatively, PAR-6–aPKC complexes can

interact with Lgl, which interacts with syntaxins and myosin.

NATURE IMMUNOLOGY VOLUME 7 NUMBER 11 NOVEMBER 2006 1145


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© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

a

b

c

Arrest and cytoskeletal relaxation

Assembly of microclusters and polarity modulators

TCR

Dlg

Scrib

Crumbs

Active transport?

TCR

TCR-Zap-70

CD4–Lck

Scrib–βPIX– ?

PAK

Crumbs– ? Pals1– PAR-6–aPKC

in the central-supramolecular activation cluster (c-SMAC), during the first

minutes of contact 17,47,48,55 , is probably another participant in this dynamic

process. Dlg associates directly with the Src homology 3 domains of both

the Zap70 and Lck tyrosine kinases 48,55 . In that way, Dlg probably senses

the proximity between Lck and Zap70 that occurs during clustering of

TCR and CD4 proteins and might even promote further aggregation of

those proteins (Fig. 3b). Dlg also interacts with microtubule kinesins 56 and

thus may act to initiate repolarization of the microtubule-based cytoskeleton.

Finally, the Lck-Zap70-Dlg complex also seems to include WASP 48 ,

and formation of that complex may help to move WASP into proximity

of active Cdc42. Transient phosphorylation of Zap70 and Lck when the

TCR is activated may trigger conformational changes in that complex and

promote its alteration from a motile to an IS form. Loss of Dlg from the

IS at later times 17 is consistent with loss of active Lck in the late IS 57 . It is

perhaps notable that elimination of Dlg by means of short hairpin RNA in

cell lines can alternatively augment 47 or attenuate 48 T cell activation. Those

observations are perhaps indicative of the importance of polarity in making

T cells receptive to signals in the first place or, alternatively, in stabilizing the

newly emerging signal, and those disparate results may reflect the polarized

nature of the stimulation provided in the various experiments.

Like Dlg, Scrib moves from the uropod, is transiently localized in the

nascent IS and subsequently moves to a position mainly outside the IS 17 . In

TCR

TCR

CD28

LFA-1

Rac

Ca 2+

Lat

SLP-76

PI(3)K

Kinesin?

cpERM

ERM

MyoII (filamentous)

P MyoII (hexameric)

WASP TCR-actin complexes

PAK

WASP

Dlg

Gakin (kinesin)

Unconventional myosin

Figure 3 Early events in polarity modulation. Specific multiprotein

complexes are suggested to mediate changes in polarity and morphology.

(a) Cytoskeletal relaxation is thought to occur as a result of cpERM

dephosphorylation as well as debundling of myosin II (MyoII) filaments.

(b) Specific multiprotein complexes then assemble at the T cell–APC

membrane contact site. These include TCR-actin complexes, Scrib

complexes (perhaps containing βPIX and PAK), TCR-Dlg complexes (perhaps

nucleating actin) and crumbs complexes (perhaps recruiting Pals and PAR-6

complexes). Molecules in gray italics have not yet been assessed in T cells,

but all others have been localized as presented here. All interactions are

not always presented here (for example, Zap70 has been linked to WASP

through multiple pathways). (c) Active transport is suggested to complete

repolarization. Myosin motors may be instrumental in recruiting uropodal

components along the membrane or through the cell. Similarly, microtubulebased

motors may recruit molecules toward or within the IS. PI(3)K,

phosphatidylinositol-3-OH kinase.

the IS, Scrib may function to recruit the Rac and Cdc42 guanine-exchange

factor βPIX (Fig. 3b). That protein has been shown to recruit PAK to the

IS in T cells 58 . Therefore, defects in Scrib-deficient T cells 17 and in T cells

expressing dominant negative PAK 59 may reflect a requirement for a ScribβPIX-PAK

complex during T cell activation.

Finally, the regulation of PAR-6-based cassettes may represent a more

complex mechanism of influencing local polarity in response to signaling.

The PAR-6–aPKC heterodimer can bind several alternative partners

through its PDZ domain, including Lgl, PAR-3 and Pals1 (protein associated

with Lin7) 60 . In eukaryotes, Pals1 is recruited to the cell surface by

crumbs, the mammalian homolog of Lin7, and functions to recruit the

PAR-6–aPKC complex to the cell cortex. In the case of T cell IS, crumbs1

is constitutively localized in the IS 17 and thus may lend ‘apical identity’ to

this surface (Fig. 3b). The mechanism by which crumbs is localized to the

IS, however, is yet to be determined.

Release of cytoskeletal tension (Fig. 3a) and assembly of polarity complexes

(Fig. 3b) are probably supplemented by an active transport mechanism

that moves proteins from the former uropod into the IS (Fig. 3c).

Surface movements exceeding the rate of diffusion have been noted for

TCRs and for beads moving along the cell surface 61 and have been shown

to depend on phosphatidylinositol-3-OH kinase and myosin 3 . Conversely,

movement of SLP-76 into the central IS seems to rely on microtubules 62 ,

suggesting that a microtubule-based movement, perhaps mediated by a

kinesin, also functions to reorient critical signaling proteins.

Stabilization of polarity in the IS

In a stable IS, actin and actomyosin contraction is diverted away from

rapid motility and functions in maintaining the contact. In that configuration,

the bulk of TCRs and many other receptors are polarized toward the

IS 3,10,61,63,64 . However, a second pool of those receptors often accumulates

at the opposite end of the cell, in what is referred to as the ‘distal pole

complex’ 65 . Once established, that asymmetric distribution is apparently

stable for many hours.

Despite being relatively nonmotile, engaged lymphocytes continuously

expand and contract away from the c-SMAC in the peripheral SMAC

(p-SMAC) and distal SMAC (d-SMAC) regions, where new peptide–major

histocompatibility complex ligands might be encountered and where

microclusters of TCRs continue to form 51,66 . One likely model proposes

that actin fibers initiating at or beyond microclusters in the p-SMAC elongate

into the c-SMAC, perhaps as a result of WASP activation ‘downstream’

of TCR signals 67 (Fig. 4a). Engaged integrins in the p-SMAC might also

promote activation of Cdc42 and WASP, as well as of aPKC, as they do

in migrating astrocytes 68 . The elongating actin fibers might encounter

resistance both from elements in the p-SMAC (generating a retrograde

direction of movement that ultimately returns to the c-SMAC) and from

pSMAC-localized myosin motors (which ‘tug’ on fibers, pulling them backward

into the c-SMAC). A selective ‘clutch’ mechanism, perhaps provided

by CD2-associated protein, which has been associated with TCR internalization

69 , may allow microclusters to attach to this moving ‘radial fiber’ and

become localized in the central IS.

Integrins and the microtubule cytoskeleton are also repolarized after

TCR engagement, and the MTOC faces the IS (Fig. 4b,c). The general

mechanisms underlying MTOC movement are complicated and probably

vary among cell types. For instance, in 3T3 fibroblasts initiating movement

into a wound, the MTOC actually remains stationary while the nucleus is

pushed backward by retrograde actin flow, thereby placing the MTOC in

front of the nucleus. PAR-6 and aPKC are needed to hold the MTOC in

position, whereas Cdc42, myotonic dystrophy–related kinase and myosin

function are required for nuclear relocation 70 . Conversely, in astrocytes, the

MTOC is actively pulled forward by microtubules by a mechanism that

depends on Cdc42, PAR-6–aPKC and dynein and that involves capture of

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a b c d e

Actomyosin cytoskeleton

Microtubule cytoskeleton Surface proteins Vesicle traffic

Polarity proteins

+

+

+

cpERMs

Membrane

recycling

vesicles

?

Dlg Scrib

Lgl? lgl?

cpERMs

Crb

© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

Distal

pole

Loose

filaments

+

+

+

Rapid

cycling?

ERM-bound receptors

Engaged receptors

Adhesive integrins

the microtubule ‘plus’ ends at the leading edge of the cell by adenomatosis

polyposis coli protein and the polarity protein Dlg1 (refs. 68,71,72). The

mechanism of MTOC reorientation in T cells remains to be elucidated.

cpERMs

Secretory vesicles

Figure 4 Five polarity systems in established T cell couples. As in Figure 1, polarity can be viewed from the perspective of five different types of events.

(a,b) The hypothesized ‘radial fibers’ of the actin cytoskeleton (a) and tubulin cytoskeleton (b) represent the profound changes in cytoskeletal reorganization

that occur. (c) Integrins and signaling receptors are ultimately arrayed mainly in the IS as overlapping densities. (d) Many types of vesicles are transported

toward the IS, and we suggest the existence of an additional recycling pool at or near the c-SMAC. (e) Polarity proteins are arrayed in patterns that

presumably reinforce IS stability.

Dlg Scrib

Polarized endocytosis and exocytosis in the IS

Controlled internalization and delivery of vesicles between the cytoplasm

and the plasma membrane is a key component regulating the polarity and

function of motile as well as engaged T cells. For example, in cytotoxic

T lymphocytes, secretory vesicles containing cytotoxic granules line up

alongside the TCR in the IS 73 . Secretory vesicles bearing the cytokines

interleukin 2 and interferon-γ also line up facing the APC, although others

(such as those containing tumor necrosis factor) do not demonstrate

obvious directional ‘preferences’ 11 . Secretory vesicles bearing interleukin 2

and interferon-γ localize together with vesicles defined by Rab proteins as

well as by the v-SNARE syntaxin 6 (ref. 11).

Vesicular release is probably also important for the reinforcement or

modulation of signaling at the IS. Studies of Lck 74 , Lat 75 , TCR 10 and

CTLA-4 (ref. 76) has shown that vesicles bearing those components of the

proximal signaling cascade traffic toward the center of the IS, where they

presumably fuse with the plasma membrane and deliver vesicular content.

Cytokine receptors as well as ‘housekeeping’ proteins such as transferrin are

similarly brought to the IS 64,77 . The recycling of TCRs in vesicles may be

essential for TCR accumulation at the IS 10 , although direct movement of

the TCR into the IS along the plasma membrane 3,61 also occurs. SNAP-23

and syntaxin 4, both of which are components of t-SNAREs, are enriched

in plasma membrane regions at or near the TCR accumulation in the IS,

whereas vesicles bearing cellubrevin and v-SNAREs localize on vesicles

inside the cytoplasm, juxtaposed to the IS 10 . In summary, these observations

suggest the existence of an exocytic machine that delivers proteins

into the IS.

Perhaps equally important to exocytosis is the uptake of proteins by

endocytosis in regions adjacent to or in the IS. It is believed that internalization

occurs at the center of the IS to facilitate TCR degradation 69,78 .

Alterations in CD2-associated protein, for example, result in decreased

TCR internalization 69,79 , and this protein has been associated with ubiquitin

E3 ligases such as Cbl. However, more than simple degradation may

be occurring at the IS. Continuous ruffling of membrane in the peripheral

region as well as coalescing flow toward the central zone may require

consistent reintroduction of membrane at the far outer domains. We thus

suggest that a vesicular cycle (Fig. 4d) may serve to recycle proteins through

the IS during serial receptor-ligand engagements.

How does exocytosis coordinate with the other polarity participants

present in the established IS? Lgl associates with syntaxin-4 in mammalian

cells 80 and may therefore influence the placement of t-SNARE on the

plasma membrane in the peripheral IS, into which proteins may be recycled

(Fig. 4e). Lgl protein may associate with cellular myosins, and such an interaction

may influence the placement of adjacent t-SNAREs and thereby also

influence the adhesiveness of integrins. Different t-SNAREs are required

for the sorting of vesicles to distinct regions of the plasma membrane.

For example, syntaxin-4 is involved in apical sorting, whereas syntaxin-3

is necessary for basolateral sorting. Distinct Rab proteins have also been

associated with that. For example, Rab17 seems to be required specifically

for apical sorting 81 . Rab13 has a specialized function in the recycling and

delivery of transmembrane proteins that form the tight junctions of epithelial

cells, such as occludin 82 . In cytotoxic T lymphocytes, the delivery of lytic

granules to the IS depends on Rab27a, which is important for regulated

secretion in several cell types 73 , and IS-localized secretory vesicles containing

interleukin 2 are associated with Rab3d and Rab19 (ref. 11). Although

syntaxin-4 accumulates along the IS near the TCR 10 , the specialized Rab

proteins involved in recycling membranes at the IS are not yet known.

Future perspectives

Although re-establishing polarity during signaling remains a focus of ongoing

work in this area, there is yet another arena in which polarity and its

modification is likely to be potentially more clinically relevant. Underlying

normal T cell surveillance is presumably a coordinated series of cues for

the T cell to follow. Although the organization of those cues remains to

be completely elucidated, it is certain that chemokine gradients into and

throughout organs serve to orient T cell polarity (Fig. 1). The presence

and density of integrin substrates provides a potential ‘highway’ on which

T cells might ‘crawl’. The presentation of chemokines together on an

integrin receptor–bearing surface 83 or to glycosaminoglycans on APCs 84

may also influence T cell function. Two-photon imaging studies have suggested

that T cells might travel along high endothelial venules 85 , perhaps

‘entranced’ by the unique combination of immobilized chemokines and a

local high density of adhesion molecules.

A remaining issue is whether ‘immune-privileged’ sites, such as tumors

or chronic inflammatory sites, might provide a defective series of motility

cues that accounts in part for poor T cell surveillance and tolerance to

antigens produced in these locations. Tumors, for example, co-opt both

chemokine and matrix-remodeling mechanisms that influence the chemotactic

and adhesive pathways discussed above. Might tumors create

T cell disorder by overexpressing some cues while minimizing others?

T cells are known to be much less reactive in their uropod 46 , and loss of

polarity, such as by loss of Scrib, is associated with inhibition of signaling

competence 17 . Notably, depletion of Dlg by means of short hairpin

Lgl?

NATURE IMMUNOLOGY VOLUME 7 NUMBER 11 NOVEMBER 2006 1147


REVIEW

© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

RNA results in overactive T cells in culture 47 , whereas for primary T cell

blasts stimulated with APC plus antigen, loss of Dlg attenuates activation 48 .

Perhaps the difference lies in the polarized cues that accompany the signals

received in those disparate stimulation conditions. As future experiments

examine tolerant environments in greater depth, it may be important to

consider the possibilities that a T cell might well fail to become active or

might remain tolerant precisely because of a disorganized array of conflicting

polarity cues that effectively ‘freezes’ the T cell in a state of inactivity.

ACKNOWLEDGMENTS

We thank J. Gilden for critical reading of the manuscript, and C. Lin for assistance

with graphics.

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureimmunology/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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